Site-directed Mutagenesis at the Exo III Motif of Phi 29 DNA Polymerase; Overlapping Structural Domains for the 3'-5' Exonuclease and Strand-Displacement Activities

EMBO J. 1992 Nov;11(11):4227-37.

Abstract

In this report we present the alignment of one of the most conserved segments (Exo III) of the 3'-5' exonuclease domain in 39 DNA polymerase sequences, including prokaryotic and eukaryotic enzymes. Site-directed substitutions of the two most conserved residues, which form the Exo III motif Tyr-(X)3-Asp of phi 29 DNA polymerase, did not affect single-stranded DNA binding, DNA polymerization, processivity or protein-primed initiation. In contrast, substitution of the highly conserved Tyr residue by Phe or Cys decreased the 3'-5' exonuclease activity to 7.5 and 4.1%, respectively, of the wild-type activity. Change of the highly conserved Asp residue into Ala resulted in almost complete inactivation (0.1%) of the 3'-5' exonuclease. In accordance with the contribution of the 3'-5' exonuclease to the fidelity of DNA replication, the three mutations in the Exo III motif (Y165F, Y165C and D169A) produced enzymes with an increased frequency of misinsertion and extension of DNA polymerization errors. Surprisingly, the three mutations in the Exo III motif strongly decreased (80- to 220-fold) the ability to replicate phi 29 DNA, this behaviour being due to a defect in the strand displacement activity, an intrinsic property of phi 29 DNA polymerase required for this process. Taking these results into account, we propose that the strand displacement activity of phi 29 DNA polymerase resides in the N-terminal domain, probably overlapping with the 3'-5' exonuclease active site.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Base Sequence
  • Binding Sites
  • Coliphages / enzymology*
  • Coliphages / genetics
  • DNA-Directed DNA Polymerase / genetics*
  • DNA-Directed DNA Polymerase / metabolism
  • Escherichia coli / genetics*
  • Exodeoxyribonuclease V
  • Exodeoxyribonucleases / genetics*
  • Exodeoxyribonucleases / metabolism
  • Models, Structural
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed*
  • Nucleic Acid Conformation
  • Oligodeoxyribonucleotides
  • Protein Conformation
  • Recombinant Proteins / metabolism
  • Sequence Homology, Amino Acid
  • Templates, Genetic

Substances

  • Oligodeoxyribonucleotides
  • Recombinant Proteins
  • DNA-Directed DNA Polymerase
  • Exodeoxyribonucleases
  • exodeoxyribonuclease III
  • Exodeoxyribonuclease V